The objective of this project is to elucidate through cell culture studies mechanisms of neoplastic transformation in human cells. Current emphasis is on the characterization in cultured human cells of chromatid DNA repair lesions found to be associated with malignant transformation and/or susceptibility to cancer. Since a chromatid apparently contains a single continuous DNA double strand, chromatid breaks and gaps represent unrepaired DNA strand breaks. Last year we reported that human skin fibroblasts transformed to malignant cells in culture had significantly more chromatid breaks and gaps than their normal counterparts when x-irradiated specifically during G2 phase or within 1.5 hours of metaphase. These observations, together with results from use of DNA repair inhibitors, implicated deficiencies in DNA repair in the malignant cells as we had found previously in mouse fibroblasts transformed in culture. These observations have been extended to cell lines from diverse human tumors, including sarcomas, carcinomas, glioblastomas, etc., as compared with numerous lines of normal fibroblasts. Further, skin fibroblasts from cancer prone individuals, including those with ataxia telangiectasia, Bloom's syndrome, Fanconi's anemia, Gardner's syndrome, familial polyposis or xeroderma pigmentosum show similar defects in chromatid DNA repair. It thus appears that acquisition of defects in chromatid DNA repair operative during G-2 prophase is a prerequisite, possibly the initiating step, for carcinogenesis in human cells. Another aspect of this project is the development of a transformation system with human epidermal keratinocytes for our further experimental studies. Additionally, computerized image analysis of living cells is being used to identify cytomorphologic markers of neoplastic transformation in culture.